Fabrication of Pascal‐triangle Lattice of Proteins by Inducing Ligand Strategy

A protein Pascal triangle has been constructed as new type of supramolecular architecture by using the inducing ligand strategy that we previously developed for protein assemblies. Although mathematical studies on this famous geometry have a long history, no work on such Pascal triangles fabricated from native proteins has been reported so far due to their structural complexity. In this work, by carefully tuning the specific interactions between the native protein building block WGA and the inducing ligand R‐SL , a 2D Pascal‐triangle lattice with three types of triangular voids has been assembled. Moreover, a 3D crystal structure was obtained based on the 2D Pascal triangles. The distinctive carbohydrate binding sites of WGA and the intralayer as well as interlayer dimerization of RhB was the key to facilitate nanofabrication in solution. This strategy may be applied to prepare and explore various sophisticated assemblies based on native proteins.     

Enhanced Hydrolytic Stability of Short‐Chain Poly[(R)‐3‐hydroxybutyrate] Conjugated to Native E. coli Cytoplasmic Proteins

Many prokaryotic and eukaryotic proteins are modified by post‐translational conjugation to short‐chain poly[(R)‐3‐hydroxybutyrate] (cPHB). The relative lability of ester bonds raises the concern that the cPHB may be substantially degraded by chemical hydrolysis during protein purification, thus increasing the difficulty of its detection and measurement. Here, we compare rates of acid‐ and base‐catalyzed hydrolysis of cPHB conjugated to native and denatured proteins at room temperature. E. coli cytoplasmic proteins, native or denatured by addition of guanidium hydrochloride, were treated with aqueous solutions of H₂SO₄ or NaOH at concentrations ranging from 0.1–2.0n . The loss of cPHB was measured as a function of time by a chemical assay. We find that cPHB conjugated to native proteins is surprisingly resistant to both acid‐ and base‐catalyzed hydrolysis, whereas cPHB conjugated to denatured proteins is proficiently degraded at rates proportional to acid or base concentration. The results suggest that cPHB occupies a highly protective environment within native proteins.     

An Integrated Mass Spectrometry Based Approach to Probe the Structure of the Full‐Length Wild‐Type Tetrameric p53 Tumor Suppressor

We present an integrated approach for investigating the topology of proteins through native mass spectrometry (MS) and cross‐linking/MS, which we applied to the full‐length wild‐type p53 tetramer. For the first time, the two techniques were combined in one workflow to obtain not only structural insight in the p53 tetramer, but also information on the cross‐linking efficiency and the impact of cross‐linker modification on the conformation of an intrinsically disordered protein (IDP). P53 cross‐linking was monitored by native MS and as such, our strategy serves as a quality control for different cross‐linking reagents. Our approach can be applied to the structural investigation of various protein systems, including IDPs and large protein assemblies, which are challenging to study by the conventional methods used for protein structure characterization.     

Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane‐mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co‐polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high‐resolution solid‐state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD.     

Native mass spectrometry—A valuable tool in structural biology

Proteins and the complexes they form with their ligands are the players of cellular action. Their function is directly linked with their structure making the structural analysis of protein‐ligand complexes essential. Classical techniques of structural biology include X‐ray crystallography, nuclear magnetic resonance spectroscopy and recently distinguished cryo‐electron microscopy. However, protein‐ligand complexes are often dynamic and heterogeneous and consequently challenging for these techniques. Alternative approaches are therefore needed and gained importance during the last decades. One alternative is native mass spectrometry, which is the analysis of intact protein complexes in the gas phase. To achieve this, sample preparation and instrument conditions have to be optimised. Native mass spectrometry then reveals stoichiometry, protein interactions and topology of protein assemblies. Advanced techniques such as ion mobility and high‐resolution mass spectrometry further add to the range of applications and deliver information on shape and microheterogeneity of the complexes. In this tutorial, we explain the basics of native mass spectrometry including sample requirements, instrument modifications and interpretation of native mass spectra. We further discuss the developments of native mass spectrometry and provide example spectra and applications.     

Water‐Rich Fluid Material Containing Orderly Condensed Proteins

A fluid material with high protein content (120–310 mg mL⁻¹) was formed through the ordered self‐assembly of native proteins segregated from water. This material is instantly prepared by the simple mixing of a protein solution with anionic and cationic surfactants. By changing the ratio of the surfactants based on the electrostatic characteristics of the target protein, we observed that the surfactants could function as a versatile molecular glue for protein assembly. Moreover, these protein assemblies could be disassembled back into an aqueous solution depending on the salt conditions. Owing to the water‐retaining properties of the hydrophilic part of surfactants, the proteins in this material are in a water‐rich environment, which maintains their native structure and function. The inclusion of water also provides functional extensibility to this material, as demonstrated by the preparation of an enzymatically active gel. We anticipate that the unique features of this material will permit the use of proteins not only in solution but also as elements of integrated functionalized materials.     

Chemical Synthesis of Native S‐Palmitoylated Membrane Proteins through Removable‐Backbone‐Modification‐Assisted Ser/Thr Ligation

The preparation of native S‐palmitoylated (S‐palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S‐palm membrane proteins by removable‐backbone‐modification‐assisted Ser/Thr ligation (RBM^GABA‐assisted STL). This method involves two critical steps: 1) synthesis of S‐palm peptides by a new γ‐aminobutyric acid based RBM (RBM^GABA) strategy, and 2) ligation of the S‐palm RBM‐modified peptides to give the desired S‐palm product by the STL method. The utility of the RBM^GABA‐assisted STL method was demonstrated by the synthesis of rabbit S‐palm sarcolipin (SLN) and S‐palm matrix‐2 (M2) ion channel. The synthesis of S‐palm membrane proteins highlights the importance of developing non‐NCL methods for chemical protein synthesis.     

Self‐Assembly of Proteins: Towards Supramolecular Materials

The study of protein self‐assembly has attracted great interest over the decades, due to the important role that proteins play in life. In contrast to the major achievements that have been made in the fields of DNA origami, RNA, and synthetic peptides, methods for the design of self‐assembling proteins have progressed more slowly. This Concept article provides a brief overview of studies on native protein and artificial scaffold assemblies and highlights advances in designing self‐assembling proteins. The discussions are focused on design strategies for self‐assembling proteins, including protein fusion, chemical conjugation, supramolecular, and computational‐aided de novo design.     

A Double Cation–π‐Driven Strategy Enabling Two‐Dimensional Supramolecular Polymers as Efficient Catalyst Carriers

The cation–π interaction is a strong non‐covalent interaction that can be used to prepare high‐strength, stable supramolecular materials. However, because the molecular plane of a cation‐containing group and that of aromatic structure are usually perpendicular when forming a cation–π complex, it is difficult to exploit the cation–π interaction to prepare a 2D self‐assembly in which the molecular plane of all the building blocks are parallel. Herein, a double cation–π‐driven strategy is proposed to overcome this difficulty and have prepared 2D self‐assemblies with long‐range ordered molecular hollow hexagons. The double cation–π interaction makes the 2D self‐assemblies stable. The 2D self‐assemblies are to be an effective carrier that can eliminate metal‐nanoparticle aggregation. Such 2D assembly/palladium nanoparticle hybrids are shown to exhibit recyclability and superior catalytic activity for a model reaction.     

Polyphenol‐Mediated Assembly of Proteins for Engineering Functional Materials

Functional materials composed of proteins have attracted much interest owing to the inherent and diverse functionality of proteins. However, establishing general techniques for assembling proteins into nanomaterials is challenging owing to the complex physicochemical nature and potential denaturation of proteins. Here, a simple, versatile strategy is introduced to fabricate functional protein assemblies through the interfacial assembly of proteins and polyphenols (e.g., tannic acid) on various substrates (organic, inorganic, and biological). The dominant interactions (hydrogen‐bonding, hydrophobic, and ionic) between the proteins and tannic acid were elucidated; most proteins undergo multiple noncovalent stabilizing interactions with polyphenols, which can be used to engineer responsiveness into the assemblies. The proteins retain their structure and function within the assemblies, thereby enabling their use in various applications (e.g., catalysis, fluorescence imaging, and cell targeting).     

Synthesis of l‐ and d‐Ubiquitin by One‐Pot Ligation and Metal‐Free Desulfurization

Native chemical ligation combined with desulfurization has become a powerful strategy for the chemical synthesis of proteins. Here we describe the use of a new thiol additive, methyl thioglycolate, to accomplish one‐pot native chemical ligation and metal‐free desulfurization for chemical protein synthesis. This one‐pot strategy was used to prepare ubiquitin from two or three peptide segments. Circular dichroism spectroscopy and racemic protein X‐ray crystallography confirmed the correct folding of ubiquitin. Our results demonstrate that proteins synthesized chemically by streamlined 9‐fluorenylmethoxycarbonyl (Fmoc) solid‐phase peptide synthesis coupled with a one‐pot ligation–desulfurization strategy can supply useful molecules with sufficient purity for crystallographic studies.     

Mitochondria‐Targeting,Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small‐molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS–NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell‐penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione‐triggered biodegradation and of native, functional proteins into the mitochondria.     

Mitochondria‐Targeting,Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small‐molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS–NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell‐penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione‐triggered biodegradation and of native, functional proteins into the mitochondria.     

Imidazole‐Aided Native Chemical Ligation: Imidazole as a One‐Pot Desulfurization‐Amenable Non‐Thiol‐Type Alternative to 4‐Mercaptophenylacetic Acid

Various bioactive proteins have been synthesized by native chemical ligation (NCL) and its combination with subsequent desulfurization (e.g., conversion from Cys to Ala). In NCL, excess 4‐mercaptophenylacetic acid (MPAA) is generally added to facilitate the reaction. However, co‐elution of MPAA with the ligation product during preparative high‐performance liquid chromatography sometimes reduces its usefulness. In addition, contamination of MPAA disturbs subsequent desulfurization. Here, we report for the first time that imidazole can be adopted as an alternative to MPAA in NCL using a peptide‐alkylthioester. The efficiency of the imidazole‐aided NCL (Im‐NCL) is similar to that of traditional MPAA‐aided NCL. As model cases, we successfully synthesized adiponectin(19‐107) and [Ser(PO₃H₂)⁶⁵]‐ubiquitin using Im‐NCL with a one‐pot desulfurization.     

Structural Studies of Self‐Assembled Subviral Particles: Combining Cell‐Free Expression with 110 kHz MAS NMR Spectroscopy

Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native‐like lipid environment. Cell‐free protein synthesis and solid‐state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) can be produced by cell‐free expression, and that the protein self‐assembles into subviral particles. Proton‐detected 2D NMR spectra recorded at a magic‐angle‐spinning frequency of 110 kHz on <500 μg protein show a number of isolated peaks with line widths comparable to those of model membrane proteins, paving the way for structural studies of this protein that is homologous to a potential drug target in HBV infection.     

A Multivalent Structure‐Specific RNA Binder with Extremely Stable Target Binding but Reduced Interaction with Nonspecific RNAs

By greatly enhancing binding affinities against target biomolecules, multivalent interactions provide an attractive strategy for biosensing. However, there is also a major concern for increased binding to nonspecific targets by multivalent binding. A range of charge‐engineered probes of a structure‐specific RNA binding protein PAZ as well as multivalent forms of these PAZ probes were constructed by using diverse multivalent avidin proteins (2‐mer, 4‐mer, and 24‐mer). Increased valency vastly enhanced the binding stability of PAZ to structured target RNA. Surprisingly, nonspecific RNA binding of multivalent PAZ can be reduced even below that of the PAZ monomer by controlling negative charges on both PAZ and multivalent avidin scaffolds. The optimized 24‐meric PAZ showed nearly irreversible binding to target RNA with negligible binding to nonspecific RNA, and this ultra‐specific 24‐meric PAZ probe allowed SERS detection of intact microRNAs at an attomolar level.     

Charge‐Induced Secondary Structure Transformation of Amyloid‐Derived Dipeptide Assemblies from β‐Sheet to α‐Helix

Secondary structures such as α‐helix and β‐sheet are the major structural motifs within the three‐dimensional geometry of proteins. Therefore, structure transitions from β‐sheet to α‐helix not only can serve as an effective strategy for the therapy of neurological diseases through the inhibition of β‐sheet aggregation but also extend the application of α‐helix fibrils in biomedicine. Herein, we present a charge‐induced secondary structure transition of amyloid‐derived dipeptide assemblies from β‐sheet to α‐helix. We unravel that the electrostatic (charge) repulsion between the C‐terminal charges of the dipeptide molecules are responsible for the conversion of the secondary structure. This finding provides a new perspective to understanding the secondary structure formation and transformation in the supramolecular organization and life activity.     

Oxetane Grafts Installed Site‐Selectively on Native Disulfides to Enhance Protein Stability and Activity In Vivo

A four‐membered oxygen ring (oxetane) can be readily grafted into native peptides and proteins through site‐selective bis‐alkylation of cysteine residues present as disulfides under mild and biocompatible conditions. The selective installation of the oxetane graft enhances stability and activity, as demonstrated for a range of biologically relevant cyclic peptides, including somatostatin, proteins, and antibodies, such as a Fab arm of the antibody Herceptin and a designed antibody DesAb‐Aβ against the human Amyloid‐β peptide. Oxetane grafting of the genetically detoxified diphtheria toxin CRM₁₉₇ improves significantly the immunogenicity of this protein in mice, which illustrates the general utility of this strategy to modulate the stability and biological activity of therapeutic proteins containing disulfides in their structures.     

Poly(arginine)‐Selective Coprecipitation Properties of Self‐Assembling Apoferritin and Its Tb3+ Complex: A New Luminescent Biotool for Sensing of Poly(arginine) and Its Protein Conjugates

The apoferritin protein and apoferritin–Tb³⁺ complex were demonstrated to form oligomeric and polymeric self‐assemblies in neutral aqueous solutions, based on characterization by using luminescence and UV/Vis spectroscopy, dynamic light scattering, and transmission electron microscopy. Addition of a 20‐mer or higher poly(arginine) to the solution resulted in coprecipitation through nanoscale interactions, while biological proteins and other poly(amino acids) rarely yielded precipitates under the conditions employed. The apoferritin–Tb³⁺ complex assembly exhibited a particularly long‐lived green luminescence in aqueous solution, and its poly(arginine)‐selective precipitation behavior was followed by monitoring the changes in luminescence. The poly(arginine)‐tagged albumin precipitated selectively and quantitatively, so that the apoferritin–Tb³⁺ complex can function as a new luminescent biotool for the sensing of poly(arginine) and its protein conjugates.     

A Convenient and Versatile Amino‐Acid‐Boosted Biomimetic Strategy for the Nondestructive Encapsulation of Biomacromolecules within Metal–Organic Frameworks

Herein, an amino‐acid‐boosted biomimetic strategy is reported that enabled the rapid encapsulation, or co‐encapsulation, of a broad range of proteins into microporous metal–organic frameworks (MOFs), with an ultrahigh loading efficiency. It relies on the accelerated formation of prenucleation clusters around proteins via a metallothionein‐like self‐assembly. The encapsulated proteins maintained their native conformations, and the structural confinement within porous MOFs endowed enzymes with excellent bioactivity, even in harsh conditions (e.g. in the presence of proteolytic or chemical agents or at high temperature). Furthermore, owing to the merits of nondestructive and protein surface charge‐independent encapsulation, the feasibility of this biomimetic strategy for biostorage, enzyme cascades, and biosensing was also verified. It is believed that this convenient and versatile encapsulation strategy has great promise in the important fields of biomedicine, catalysis, and biosensing.